Vertebrate nonmuscle and smooth muscle myosins are regulated by phosphorylation of the 20 kDa light chains. However, the heavy chains of these myosins can also be phosphorylated. Our present goal is to understand the function of smooth muscle myosin heavy chain phosphorylation. We began by identifying the kinase(s) that phosphorylate smooth muscle myosin heavy chains in intact vascular cells. Of a variety of serine/threonine kinases assayed, only casein kinase II and calcium/calmodulin-dependent protein kinase II phosphorylated the smooth muscle myosin heavy chain to a significant extent in vitro. Two-dimensional maps of tryptic peptides derived from heavy chains phosphorylated in cultured vascular cells revealed one major and one minor phosphopeptide. Identical tryptic peptide maps were obtained from heavy chains phosphorylated in vitro with casein kinase II, but not with calcium/calmodulin-dependent protein kinase II. Of note, the 204 kDa smooth muscle myosin heavy chain, but not the 200 kDa heavy chain isoform, was phosphorylated by casein kinase II. Partial sequence of the tryptic phosphopeptides generated following phosphorylation by casein kinase II yielded the following: VIENADGS*EEEV. The S* represents the Ser(PO4) which is in an acidic environment, as is typical for casein kinase II phosphorylation sites. By comparison with the deduced amino acid sequence for rabbit uterine smooth muscle myosin (Nagai, R., Kuro-o, M., Babij, P., and Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737), we have localized the phosphorylated serine residue to the non-helical tail of the 204 kDa isoform of the smooth muscle myosin heavy chain. The ability of the 204 kDa isoform, but not the 200 kDa isoform, to serve as a substrate for casein kinase II suggests that these two isoforms can be differentially regulated.